The localization of two epitopes recognized by the monoclonal antibody PCG-4 on toxin A of Clostridium difficile

Clostridium difficile causes pseudomenbranous colitis (PMC) and diarrhea in humans. Toxigenic strains of C. difficile produce two toxins. Toxin A is an enterotoxin and cytotoxin, and toxin B is a potent cytotoxin. The gene encoding toxin A has been sequenced and was shown to possess a 2.5 kb region, containing 38 similar repeating amino acid sequences, at the 3' -end of the gene. This region of the toxin A gene codes for the carbohydrate-binding portion of the toxin. The monoclonal antibody PCG-4 (MAb) binds to this portion of toxin A and neutralizes its enterotoxic activity. In addition, this monoclonal antibody has been shown to immunoprecipitate toxin A, suggesting that the MAb PCG-4 is binding to two or more similar epitopes on the toxin. The goal of this research project was to identify the neutralizing epitopes recognized by the MAb PCG-4 on the surface of the toxin A. To map the epitopes bound by the MAb PCG-4, a series of overlapping deletion clones were constructed from a 4.7 kb fragment from the 3'-end of the toxin A gene. The recombinant polypeptides expressed by these clones were tested for reactivity with the MAb PCG-4. By comparing the overlapping polypeptides, defined as either PCG-4 reactive or nonreactive, I localized the PCG-4 epitope to a 44-amino acid sequence situated between the amino acid residues 2098-2141 of toxin A A similarity search of the toxin with the 44-amino acid sequence containing the PCG-4 epitope revealed the presence of two other possible PCG-4 epitopes located between the amino acid residues 2355-2398 and 2459-2502. However, subsequent cloning experiments showed that only the region located between the amino acid residues 2355-2398 contained a PCG-4 reactive epitope. The identification of two similar epitopes within the toxin's structure explains how this monoclonal antibody is able to immunoprecipitate toxin A in the absence of subunits. Furthermore, I found that small recombinant polypeptides, containing the PCG-4 epitope lost reactivity with this monoclonal antibody following denaturation, suggesting that the epitopes recognized by this monoclonal antibody are conformationally dependent.

Name thesis:degree_name

Master of Science

Level thesis:degree_level

masters

Discipline thesis:degree_discipline

Microbiology

Department dc:contributor.department

Microbiology

Grantor dc:publisher

Virginia Tech

Year dc:date.issued

1990

Author dc:creator

• Frey, Steven M.

Chair dc:contributor.committeechair

• Wilkins, Tracy D.

Committee members dc:contributor.committeemember

• Johnson, John L.
• Chen, Jiann-Shin

Statement dc:rights

• In Copyright

Licence dc:rights.uri

• http://rightsstatements.org/vocab/InC/1.0/

Language dc:language.iso

en

Dc Identifier Other

etd-05022009-040613

OAI identifier oai:identifier

oai:vtechworks.lib.vt.edu:10919/42409